Part:BBa_K4273017

pTDH3-DDGS-tTDH1-pPGK1-OMT-tPGK1

The first and second gene of gene clusters involved in biosynthesis of shinorine in cyanobacteria N. punctiforme.

encoding 2-demethyl 4-deoxygadusol synthase (DDGS) that converts sedoheptulose 7-phosphate (S7P) to 2-demethyl-4-deoxygadusol (DDG) and O-methyltransferase (O-MT) that converts 2-demethyl-4-deoxygadusol (DDG) to 4-deoxygadusol (4-DG)

Usage and Biology

Figure 1: Insertion of DDGS and OMT at 308 position. By transforming CRISPR-308 plasmid pCRCT-308, LA, DDGS, OMT and RA, the two genes should be inserted at 308 position (A). We expanded the homogenous arms, DDGS and OMT genes through PCR and transformed them into L2 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-DDGS and OMT (C) and verified this result through the sequencing testing (D), obtaining the L3 strain.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 3270
  Illegal XhoI site found at 1922
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 2168
  Illegal BsaI.rc site found at 3280
  

Functional Parameters

Figure 2: Optimizing production by inserting OMT-DDGS and deleting Nqm1 gene. Nqm1 function similarly as TAL1, which reduces S7P concentrations. By transforming CRISPR-Nqm1 plasmid pCRCT-Nqm1, LA, OMT, DDGS, and RA, the two genes should be inserted at Nqm1 position; we switched the promoter of DDGS to pPGK1 and OMT to pTDH3 in order to have equal net transcription rate of DDGS and OMT genes. We expanded the homogenous arms, OMT, DDGS genes through PCR and transformed them into L5 strains for it to be assembled in the genome. We performed colony PCR on the yeast colonies to determine the existence of LA-OMT, and DDGS (C) and verified this result through the sequencing testing (D), obtaining the L6 strain.

Parameters

Figure 3: Shinorine and porphyra-334 production after optimization. We transformed porphyra-334 producing plasmid Np5598-NlmysD and shinorine producing plasmid Np5598-Np5597 into the L3 and L6 strains (A) and compared the absorption spectrum after 72 hours of fermentation. The absorption peak at 334nm of L7 strains displayed significant improvement compared to L5 strains (B) . From OD334, we concluded that in comparison with the L5 assembly, porphyra-334 production in L6 yeast increased by 91.8%, and shinorine production increased by 70.9% (C).